Polypeptide formulation

ABSTRACT

The present invention relates to an aqueous pharmaceutical composition suitable for long-term storage of polypeptides containing an Fc domain of an immunoglobulin, methods of manufacture, methods of administration and kits containing same.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/360,257, filed Feb. 27, 2002, which is herebyincorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to an aqueous pharmaceuticalcomposition suitable for long-term storage of polypeptides containing anFc domain of an immunoglobulin, methods of manufacture of thecomposition, methods of administration and kits containing same.

BACKGROUND

[0003] After production, polypeptides must typically be stored prior totheir use. Frequently, when stored for extended periods polypeptides areunstable in solution (Manning et al., 1989, Pharm. Res. 6:903-918).Accordingly, additional processing steps have been developed to allowfor a longer shelf life including drying, e.g., lyophilization. However,lyophilized pharmaceutical compositions are less convenient for the enduser.

[0004] Typical practices to improve polypeptide stability can beaddressed by varying the concentration of elements with the formulation,or by adding excipients to modify the formulation (U.S. Pat. Nos.5,580,856 and 6,171,586). The use of additives, while improving storage,can still results in inactive polypeptides. In addition, in the case oflyophilization, the rehydration step can introduce conditions thatresult in inactivation of the polypeptide by, for example, aggregationor denaturation (Hora et al., 1992, Pharm. Res., 9:33-36; Liu et al.,1991, Biotechnol. Bioeng., 37:177-184). In fact, aggregation ofpolypeptides is undesirable as it may result in immunogenicity (Clelandet al., 1993, Crit. Rev. Therapeutic Drug Carrier Systems, 10:307-377;and Robbins et al., 1987, Diabetes, 36:838-845).

[0005] The present invention addresses these issues by providing a novelstable liquid formulation that allows long term storage of a polypeptidecontaining an Fc domain of an immunoglobulin.

SUMMARY

[0006] The invention relates, in part, to a stable aqueouspharmaceutical composition comprising a therapeutically effective amountof an Fc domain containing polypeptide, an aggregation inhibitorselected from the group consisting of L-arginine and L-cysteine.Optionally, the composition can include a buffer, a tonicity modifierand one or more excipients. In one aspect, the buffer maintains thecomposition pH at a range of about 6.0 and about 7.0. Preferably, the Fcdomain containing polypeptide is stable in the present formulation forat least three months at 2-8° C. and/or is stable following one or morefreezing and thawing cycles of the formulation.

[0007] The invention also relates to a method of formulating apharmaceutical composition, the composition an Fc domain containingpolypeptide with an aggregation inhibitor selected from the groupconsisting of L-arginine and L-cysteine. Optionally, one can also add tothe pharmaceutical composition a buffer, a tonicity modifier and/or anexcipient. In one aspect, the pharmaceutical composition is formulatedat a pH range between pH 6.0 and 7.0.

[0008] The invention also relates to a method of treating a mammalcomprising administering a therapeutically effective amount of thepharmaceutical composition described herein, wherein the mammal has adisease or disorder that can be beneficially treated with a Fc domaincontaining polypeptide in the composition.

[0009] The invention also relates to a method of accelerated stabilitytesting of an Fc domain containing polypeptide in a pharmaceuticalcomposition of the invention comprising the steps of storing thecomposition at 37° C. for one month and measuring the stability of thepolypeptide.

[0010] In another embodiment, the present invention is directed to a kitor container, which contains an aqueous pharmaceutical composition ofthe invention. The kit can also be accompanied by instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1: Size exclusion chromatography (SEC) data for lots A, B, Cand D, and lot 1 stored for up to 1 year at 2-8° C.

[0012]FIG. 2: SEC data for lots A, B, C and D, and lot 1 stored for upto 1 year at 37° C.

[0013]FIG. 3: Denatured SEC (dSEC) data for lots A, B, C and D, and lot1 stored for up to 1 year at 2-8° C.

[0014]FIG. 4: dSEC data for lots A, B, C and D, and lot 1 stored for upto 1 year at 37° C.

[0015]FIG. 5: Hydrophobic interaction chromatography (HIC) Peak 1 andPre-peak 1 data for lots A, B, C and D, and lot 1 stored for up to 1year at 2-8° C.

[0016]FIG. 6: HIC Peak 1 and Pre-peak 1 data for lots A, B, C and D, andlot 1 stored for up to 1 year at 37° C.

[0017]FIG. 7: HIC Peak 2 data for lots A, B, C and D, and lot 1 storedfor up to 1 year at 2-8° C.

[0018]FIG. 8: HIC Peak 2 data for lots A, B, C and D, and lot 1 storedfor up to 1 year at 37° C.

[0019]FIG. 9: HIC Peak 3 data for lots A, B, C and D, and lot 1 storedfor up to 1 year at 2-8° C.

[0020]FIG. 10: HIC Peak 3 data for lots A, B, C and D, and lot 1 storedfor up to 1 year at 37° C.

[0021]FIG. 11: Binding activity of lots A, B, C and D, and lot 1 storedfor 12 months at −70, 2-8, 30, and 37° C.

[0022]FIG. 12: Bioactivity of lots A, B, C and D, and lot 1 stored for12 months at −70, 2-8, 30, and 37° C.

DETAILED DESCRIPTION

[0023] In long-term storage of pharmaceutical compositions containingpolypeptides, including aqueous and lyophilized formulations, activepolypeptides can be lost due to aggregation and/or degradation. Thus,the present invention is directed to an aqueous formulation thatsurprisingly allows for stable long-term storage of a pharmaceuticalcomposition wherein the active ingredient in the composition is apolypeptide having an Fc domain of an antibody. This formulation isuseful, in part, because it is more convenient to use for the patient,as this formulation does not require any extra steps such asrehydrating.

[0024] As used herein, the phrase “pharmaceutical composition” isunderstood to refer to a formulation comprised of a polypeptide preparedsuch that it is suitable for injection and/or administration into apatient in need thereof. More particularly, a pharmaceutical compositionis substantially sterile and does not contain any agents that are undulytoxic or infectious to the recipient. Further, it is to be understoodthat, as used herein, a solution or liquid formulation is meant to meana liquid preparation that contains one or more chemical substancesdissolved in a suitable solvent or mixture of mutually misciblesolvents.

[0025] In addition, as used herein, the term “about” is understood tomean that there can be variation in the concentration of a component ofthe described formulation that can be to 5%, 10%, 15% or up to andincluding 20% of the given value. For example, if a formulation hasabout 10 mg/ml of an Fc domain containing polypeptide, this isunderstood to mean that a formulation can have between 8 to 12 mg/ml ofthe stated polypeptide.

[0026] In one embodiment, the formulation is comprised of an Fc domaincontaining polypeptide, an aggregation inhibitor selected from groupconsisting of L-arginine and L-cysteine, and, optionally, a buffer, atonicity modifier and additional excipients as necessary. L-arginine hasbeen used to assist refolding of insoluble polypeptides, particularlythose expressed to high levels in inclusion bodies in bacteria. However,L-arginine has not been utilized successfully to enhance stability of Fcdomain containing polypeptides in pharmaceutical compositions (Soejimaet al., 2001, J. Biochem., 130:369-277).

[0027] It is contemplated that the preparation of the composition shouldbe done in consideration of limiting injection site discomfort. It isfurther contemplated that additional active ingredients can also beincluded in the presently described composition, for example, to reduceinjection site discomfort. Such active ingredients include, but are notlimited to non-steroidal anti-inflammatory drugs such as, for example,tromethamine, in an appropriate dosage.

[0028] Polypeptides

[0029] In a particular embodiment the Fc domain containing polypeptideis a soluble form of the TNF receptor fused to an Fc domain (TNFR:Fc),however, it is to be understood that any polypeptide containing an Fcdomain is suitable for use in the instant formulation. A commerciallyavailable TNFR:Fc is known as etanercept (Enbrel®, Immunex Corporation),which is a dimeric fusion polypeptide consisting of the extracellularligand-binding portion of the human 75 kilodalton (p75) tumor necrosisfactor receptor (TNFR) linked to the Fc portion of human IgG1. The Fccomponent of etanercept contains the constant heavy 2 (CH2) domain, theconstant heavy 3 (CH3) domain and hinge region, but not the constantheavy 1 (CH1) domain of human IgG1. It is to be understood that an Fcdomain can contain one or all of the domains described above. Etanerceptis produced by recombinant DNA technology in a Chinese hamster ovary(CHO) mammalian cell expression system. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.).

[0030] Other polypeptides specifically contemplated for formulationaccording to the invention include recombinant fusion polypeptidescomprising at least a portion of an Fc domain of an antibody. Apolypeptide fused to an Fc domain and identical to or substantiallysimilar to one of the following polypeptides is suitable for use in thepresent pharmaceutical composition: a flt3 ligand, a CD40 ligand,erythropoeitin, thrombopoeitin, calcitonin, Fas ligand, ligand forreceptor activator of NF-kappa B (RANKL), tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL), thymic stroma-derivedlymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor, mast cell growthfactor, stem cell growth factor, epidermal growth factor, RANTES, growthhormone, insulin, insulinotropin, insulin-like growth factors,parathyroid hormone, interferons, nerve growth factors, glucagon,interleukins 1 through 18, colony stimulating factors, lymphotoxin-β,tumor necrosis factor (TNF), leukemia inhibitory factor, oncostatin-M,and various ligands for cell surface molecules ELK and Hek (such as theligands for eph-related kinases or LERKS).

[0031] Polypeptides suitable for formulation according to the inventionalso include recombinant fusion polypeptides comprising an Fc domain ofan antibody plus a receptor for any of the above-mentioned polypeptidesor polypeptides substantially similar to such receptors. These receptorsinclude: both forms of TNFR (referred to as p55 and p75), Interleukin-1receptors (type 1 and 2), Interleukin-4 receptor, Interleukin-15receptor, Interleukin-17 receptor, Interleukin-18 receptor,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK),receptors for TRAIL (TRAIL receptors 1, 2, 3, and 4), and receptors thatcomprise death domains, such as Fas or Apoptosis-Inducing Receptor(AIR).

[0032] Other polypeptides suitable for use in the present formulationinclude differentiation antigens (referred to as CD polypeptides) ortheir ligands or polypeptides substantially similar to either of these,which are fused to an Fc domain of an antibody. Such antigens aredisclosed in Leukocyte Typing VI (Proceedings of the VIth InternationalWorkshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan,1996). Similar CD polypeptides are disclosed in subsequent workshops.Examples of such antigens include CD27, CD30, CD39, CD40, and ligandsthereto (CD27 ligand, CD30 ligand, etc.). Several of the CD antigens aremembers of the TNF receptor family, which also includes 41BB ligand andOX40. The ligands are often members of the TNF family, as are 41BBligand and OX40 ligand. Accordingly, members of the TNF and TNFRfamilies can be formulated according to the present invention.

[0033] Enzymatically active polypeptides or their ligands can also beformulated according to the invention. Examples include recombinantfusion polypeptides comprising an Fc domain of an antibody fused to allor part of one of the following polypeptides or their ligands or apolypeptide substantially similar to one of these:metalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

[0034] The formulations and methods of the invention can also be used toprepare pharmaceutical compositions comprising antibodies, humanantibodies, humanized antibodies, chimeric antibodies, i.e. antibodieshaving human constant antibody immunoglobulin domains coupled to one ormore murine variable antibody immunoglobulin domain, and/or non-humanantibodies, or fragments thereof. Specific examples of antibodiessuitable for use in the present formulation include commerciallyavailable antibodies such as muromonab-CD3 (Orthoclone OKT-3®, OrthoBiotech), abciximab (ReoPro®, Lilly), rituximab (Rituxan®, IDEC),dacliximab (Zenapax®, Roche Laboratories), basiliximab (Simulect®,Novartis), infliximab (Remicade®, Centocor), palivizumab (Synagis®,MedImmune), trastuzumab (Herceptin®, Genentech), gemtuzuman ozogamicin(Mylotarg™, Wyeth-Ayerst), and alemtuzumab (Campath®, Berlex). Currentlyeach of the foregoing is available either as a lyophilized powderrequiring rehydration or as a concentrate requiring dilution prior toadministration. The present formulation obviates the need for anymanipulations prior to administration, e.g., rehydrating or dilution,while preserving stability of the active ingredients over long-termstorage.

[0035] The pharmaceutical composition of the invention can also be usedto store polypeptides comprising an antibody conjugated to a cytotoxicor luminescent substance. Such substances include: maytansinederivatives (such as DM1); enterotoxins (such as a Staphylococcalenterotoxins); iodine isotopes (such as iodine-125); technetium isotopes(such as Tc-99m); cyanine fluorochromes (such as Cy5.5.18); andribosome-inactivating polypeptides (such as bouganin, gelonin, orsaporin-S6).

[0036] Examples of antibodies or antibody/cytotoxin orantibody/luminophore conjugates contemplated for use in the inventioninclude those that recognize one or more of the following antigens: CD2,CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40,CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-4, IL-5, IL-8, IL-10,IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, PDGF-β,VEGF, TGF, TGF-β2, TGF-β1, EGF receptor, VEGF receptor, C5 complement,IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which isa gene product that is expressed in association with lung cancer),HER-2, a tumor-associated glycoprotein TAG-72, the SK-1 antigen,tumor-associated epitopes that are present in elevated levels in thesera of patients with colon and/or pancreatic cancer, cancer-associatedepitopes or polypeptides expressed on breast, colon, squamous cell,prostate, pancreatic, lung, and/or kidney cancer cells and/or onmelanoma, glioma, or neuroblastoma cells, TRAIL receptors 1, 2, 3 and 4,the necrotic core of a tumor, integrin alpha 4 beta 7, the integrinVLA-4, B2 integrins, TNF-α, the adhesion molecule VAP-1, epithelial celladhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3),leukointegrin adhesin, the platelet glycoprotein gp IIb/IIIa, cardiacmyosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitorof factor VIa-tissue factor), MHC I, carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is acytotoxic T lymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10beta, HLA-DR antigen, L-selectin, IFN-γ, Respiratory Syncitial Virus,human immunodeficiency virus (HIIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphylococcus aureus.

[0037] The formulations of the invention can also be used foranti-idiotypic antibodies, or substantially similar polypeptides,including but not limited to anti-idiotypic antibodies against: anantibody targeted to the tumor antigen gp72; an antibody against theganglioside GD3; or an antibody against the ganglioside GD2.

[0038] The Fc domain containing polypeptide suitable for storage in thepresent pharmaceutical composition can be produced by living host cellsthat express the polypeptide, such as hybridomas in the case ofantibodies, or host cells that that have been genetically engineered toproduce the polypeptide in the case of fusion polypeptides orantibodies. Methods of genetically engineering cells to producepolypeptides are well known in the art. See, e.g., Ausubel et al., eds.(1990), Current Protocols in Molecular Biology (Wiley, New York). Suchmethods include introducing nucleic acids that encode and allowexpression of the polypeptide into living host cells. These host cellscan be bacterial cells, fungal cells, or, preferably, animal cells grownin culture. Bacterial host cells include, but are not limited to,Escherichia coli cells. Examples of suitable E. coli strains include:HB101, DH5α, GM2929, JM109, KW251, NM538, NM539, and any E. coli strainthat fails to cleave foreign DNA. Fungal host cells that can be usedinclude, but are not limited to, Saccharomyces cerevisiae, Pichiapastoris and Aspergillus cells. A few examples of animal cell lines thatcan be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and W138. Newanimal cell lines can be established using methods well know by thoseskilled in the art (e.g., by transformation, viral infection, and/orselection). Optionally, the polypeptide can be secreted by the hostcells into the medium.

[0039] Purification of the expressed Fc domain containing polypeptidecan be performed by any standard method. When the Fc domain containingpolypeptide is produced intracellularly, the particulate debris isremoved, for example, by centrifugation or ultrafiltration. When thepolypeptide is secreted into the medium, supernatants from suchexpression systems can be first concentrated using standard polypeptideconcentration filters. Protease inhibitors can also be added to inhibitproteolysis and antibiotics can be included to prevent the growth ofmicroorganisms.

[0040] The Fc domain containing polypeptide can be purified using, forexample, hydroxyapatite chromatography, gel electrophoresis, dialysis,and affinity chromatography, and any combination of purificationtechniques known or yet to discovered. For example, protein A can beused to purify Fc domain containing polypeptides that are based on humangamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., 1983, J.Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypesand for human gamma 3 (Guss et al., 1986, EMBO J. 5:1567-1575).

[0041] Other techniques for polypeptide purification such asfractionation on an ion-exchange column, ethanol precipitation, reversephase HPLC, chromatography on silica, chromatography on heparinSEPHAROSET™, chromatography on an anion or cation exchange resin (suchas a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation can also be utilized depending on need.

[0042] Pharmaceutical Composition

[0043] The present pharmaceutical composition is prepared by combining,in addition to a purified polypeptide described above, an aggregationinhibitor. Further, a buffer, a tonicity modifier and an additionalexcipient can be added as needed. It will be understood one of ordinaryskill in the art that the combining of the various components to beincluded in the composition can be done in any appropriate order,namely, the buffer can be added first, middle or last and the tonicitymodifier can also be added first, middle or last. It is also to beunderstood by one of ordinary skill in the art that some of thesechemicals can be incompatible in certain combinations, and accordingly,are easily substituted with different chemicals that have similarproperties but are compatible in the relevant mixture.

[0044] Aggregation inhibitors reduce a polypeptide's tendency toassociate in inappropriate or unwanted ternary or quaternary complexes.Unexpectedly, the present inventors have found that the amino acidsL-arginine and/or, L-cysteine, act to reduce aggregation of Fc domaincontaining polypeptide in a formulation for long periods, e.g., twoyears or more. The concentration of the aggregation inhibitor in theformulation is preferably between about 1 mM to 1M, more preferablyabout 10 mM to about 200 mM, more preferably about 10 mM to about 100mM, even more preferably about 15 mM to about 75 mM, and yet morepreferably about 25 mM. These compounds are available from commercialsuppliers.

[0045] Buffering agents maintain pH in a desired range and variousbuffers suitable for use in the pharmaceutical composition of theinvention include histidine, potassium phosphate, sodium or potassiumcitrate, maleic acid, ammonium acetate, tris(hydroxymethyl)-aminomethane(tris), various forms of acetate and diethanolamine. One preferredbuffer is sodium phosphate as its buffering capacity is at or near pH6.2. The concentration of the buffer in the formulation is preferablybetween about 1 mM to about 1M, more preferably about 10 mM to about 200mM. Buffers are well known in the art and are manufactured by knownmethods and available from commercial suppliers.

[0046] When the pH of the pharmaceutical composition is set at or nearphysiological levels comfort of the patient upon administration ismaximized. In particular, it is preferred that the pH be within a rangeof pH about 5.8 to 8.4, with about 6.2 to 7.4 being preferred, however,it is to be understood that the pH can be adjusted as necessary tomaximize stability and solubility of the polypeptide in a particularformulation and as such, a pH outside of physiological ranges, yettolerable to the patient, is within the scope of the invention.

[0047] A tonicity modifier is understood to be a molecule thatcontributes to the osmolality of a solution. The osmolality of apharmaceutical composition is preferably regulated in order to maximizethe active ingredient's stability and also to minimize discomfort to thepatient upon administration. Where serum is approximately 300+/−50milliosmolals per kilogram. It is generally preferred that apharmaceutical composition be isotonic with serum, i.e., having the sameor similar osmolality, which is achieved by addition of a tonicitymodifier, thus it is contemplated that the osmolality will be from about180 to about 420 milliosmolals, however, it is to be understood that theosmolality can be either higher or lower as specific conditions require.Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (e.g., arginine, cysteine,histidine and glycine), salts (e.g., sodium chloride, potassium chlorideand sodium citrate) and/or saccharides (e.g., sucrose, glucose andmannitol). The concentration of the tonicity modifier in the formulationis preferably between about 1 mM to 1M, more preferably about 10 mM toabout 200 mM. Tonicity modifiers are well known in the art and aremanufactured by known methods and available from commercial suppliers.

[0048] Excipients, also referred to as chemical additives, co-solutes,or co-solvents, that stabilize the polypeptide while in solution (alsoin dried or frozen forms) can also be added to a pharmaceuticalcomposition. Examples include but are not limited to sugars/polyols suchas: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose,inositol, trehalose, glucose; polymers such as: serum albumin (bovineserum albumin (BSA), human SA or recombinant HA), dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueoussolvents such as: polyhydric alcohols, (e.g., PEG, ethylene glycol andglycerol) dimethysulfoxide (DMSO) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid;surfactants such as: Tween-80, Tween-20, SDS, polysorbate,polyoxyethylene copolymer; and miscellaneous excipients such as:potassium phosphate, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions(e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS,monolaurate, 2-O-beta-mannoglycerate or any combination of the above.

[0049] The concentration of one or more excipients in a formulation ofthe invention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent. Excipients are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

[0050] In one illustrative embodiment, a formulation of the inventioncan comprise about 25 to about 50 mg TNFR:Fc (etanercept), about 10 mMto about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate,about 0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl, atabout pH 6.0 to about pH 7.0. In another embodiment L-arginine can bereplaced with L-cysteine (at about 1 to about 500 micromolar) in theformulation. In yet another embodiment, the pH can be about pH 7.0. Inanother specific embodiment, a formulation of the invention can compriseabout 25 mg/ml TNFR:Fc, about 25 mM L-arginine, about 25 mM sodiumphosphate, about 98 mM sodium chloride, and about 1% sucrose at about pH6.2.

[0051] In another embodiment, a formulation of the invention cancomprise about 10 to about 100 mg/mL of RANK:Fc in about 10 mM to about100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate, about0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl, at aboutpH 6 to about pH 7. In a specific embodiment, the formulation of theinvention comprises 50 mg/ml RANK:Fc in about 25 mM L-arginine, about 25mM sodium phosphate, about 98 mM sodium chloride, and about 1% sucroseat about pH 6.2.

[0052] In yet another embodiment, a formulation of the invention cancomprise an effective amount of an Fc domain containing polypeptide,about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mMsodium phosphate, about 0 to 5% Mannitol and 0 to 0.2% Tween-20 at aboutpH 6 to 7. In another embodiment, a formulation of the invention cancomprise an effective amount of an antibody, such as Emab (an anti-CD22specific antibody), about 25 mM L-arginine, about 25 mM sodiumphosphate, about 4% Mannitol, about 0.02% Tween-20 and at about pH 6.0.

[0053] In yet another embodiment, the invention provides a method oftreating a mammal comprising administering a therapeutically effectiveamount of the pharmaceutical composition described herein, wherein themammal has a disease or disorder that can be beneficially treated with aFc domain containing polypeptide in the composition. In yet anotherembodiment, the Fc domain containing polypeptide is derived from thesame species of mammal as is to be treated with the composition. In aparticular embodiment, the mammal is a human patient in need oftreatment. When the Fc domain containing polypeptide of the compositionis TNFR:Fc, examples of diseases or disorders that can be treatedinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with TNFR:Fc include those described in WO 00/62790, WO01/62272 and U.S. Patent Application No. 2001/0021380, the relevantportions of which are incorporated herein by reference.

[0054] In yet another embodiment, the invention provides a method foraccelerated stability testing of the stability an Fc domain containingpolypeptide in a pharmaceutical composition of the invention comprisingthe steps of testing the activity of the polypeptide formulatedaccording to the invention prior to storage, i.e., time zero, storingthe composition at 37° C. for one month and measuring the stability ofthe polypeptide, and comparing the stability form time zero to the onemonth time point. This information is helpful for early elimination ofbatches or lots that appear to have good stability initially, yet do notstore well for longer periods.

[0055] Moreover, the present pharmaceutical composition providesimproved long-term storage such that the active ingredient, e.g., an Fcdomain containing polypeptide, is stable over the course of storageeither in liquid or frozen states. As used herein, the phrase“long-term” storage is understood to mean that the pharmaceuticalcomposition can be stored for three months or more, for six months ormore, and preferably for one year or more. Long term storage is alsounderstood to mean that the pharmaceutical composition is stored eitheras a liquid at 2-8° C. or is frozen, e.g., at −20° C. or colder. It isalso contemplated that the composition can be frozen and thawed morethan once. The term “stable” with respect to long-term storage isunderstood to mean that the active polypeptide of the pharmaceuticalcomposition does not lose more than 20%, or more preferably 15%, or evenmore preferably 10%, and most preferably 5% of its activity relative toactivity of the composition at the beginning of storage.

[0056] Effective Dose of the Pharmaceutical Composition

[0057] The appropriate dosage, or therapeutically effective amount, ofthe Fc domain containing polypeptide of the formulation will depend onthe condition to be treated, the severity of the condition, priortherapy, and the patient's clinical history and response to thetherapeutic agent. The proper dose can be adjusted according to thejudgment of the attending physician such that it can be administered tothe patient one time or over a series of administrations. Thepharmaceutical composition can be administered as a sole therapeutic orin combination with additional therapies as needed.

[0058] In one embodiment, the effective Fc domain containing polypeptideamount per adult dose ranges from about 1-500 mg/m², or from about 1-200mg/m², or from about 1-40 mg/m² or about 5-25 mg/m². Alternatively, aflat dose may be administered, whose amount may range from 2-500mg/dose, 2-100 mg/dose or from about 10-80 mg/dose. If the dose is to beadministered more than one time per week, an exemplary dose range is thesame as the foregoing described dose ranges or lower and preferablyadministered two or more times per week at a per dose range of 25-100mg/dose. In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose. The dose can be administered at biweekly, weekly doses, orseparated by several weeks (for example 2 to 8). In this example TNFR:Fc(etanercept) is generally administered at 25 mg by a single subcutaneous(SC) injection.

[0059] In many instances, an improvement in a patient's condition willbe obtained by a dose of up to about 100 mg of the pharmaceuticalcomposition one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For incurable chronic conditions theregimen may be continued indefinitely. For pediatric patients (ages4-17), a suitable regimen involves a dose of 0.4 mg/kg to 5 mg/kg of athe polypeptides of the invention, administered one or more times perweek.

[0060] In another embodiment, it is contemplated that the pharmaceuticalformulation of the invention is prepared in a bulk formulation and assuch, the components of the pharmaceutical composition are adjusted sothat it is higher than would be required for administration and dilutedappropriately prior to administration.

[0061] Administration of the Pharmaceutical Composition

[0062] The pharmaceutical compositions of this invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, and/or intrathecal. Parenteraladministration can be by bolus injection or continuous infusion.Pharmaceutical compositions for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease™, Genject™, injector pens such as GenPen™,and needleless devices such as MediJector™ and BioJector™. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

[0063] The pharmaceutical composition can also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

[0064] The pharmaceutical compositions may, if desired, be presented ina vial, pack or dispenser device which may contain one or more unitdosage forms containing the active ingredient. In one embodiment thedispenser device can comprise a syringe having a single dose of theliquid formulation ready for injection. The syringe can be accompaniedby instructions for administration.

[0065] In another embodiment, the present invention is directed to a kitor container, which contains an aqueous pharmaceutical composition ofthe invention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

[0066] The invention will be more fully understood by reference to thefollowing examples. The examples should not, however, be construed aslimiting the scope of the invention.

EXAMPLES Example 1

[0067] In order to determine the best excipient to prevent aggregationof an Fc domain containing polypeptide, TNFR:Fc was produced and testedfor light scattering of a sample (Is) containing the TNFR:Fc withvarious excipients after incubation at 51° C.+/−1° C., and compared tolight scattering of a control (Ic) sample with TNFR:Fc alone stored at2-8° C. The ratio is measured as Is/Ic, and a ratio of one represents atheoretical baseline where there is no change in the light scattering,i.e., aggregation, of the test compound. The various excipients testedincluded 5% ascorbic acid, 5% mannitol, 10% sucrose, 1%polyvinylpyrrolidone (PVP-K15), 0.1% polyethylene glycol (PEG, Mw=1000),0.6% ethanol, 1.2% glycine, 2% L-arginine, 0.01% Pluronic F68, 1.6%Betaine and 1.5% L-cysteine. Surprisingly, L-arginine was the onlyaggregation inhibitor found to keep the Is/Ic ratio below one for theentire 200 hour test period.

Example 2

[0068] TNFR:Fc produced and denoted as lots A, B, C and D were evaluatedagainst TNFR:Fc produced by a different method and having higher initialaggregation (lot 1) for stability in a liquid formulation (25 mMphosphate, 25 mM L-arginine, 98 mM NaCl, 1% sucrose, at pH 6.2) insyringes or glass vials at −70° C., −20° C., 2-8° C., 30° C., and 37° C.Samples were analyzed by size exclusion chromatography (SEC), denaturedSEC (dSEC), hydrophobic interaction chromatography (HIC), sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and forbinding and bioactivity at various timepoints. The bioactivity can bemeasured by any number of assays including by SEC, dSEC, HIC, bindingactivity and bioactivity, as discussed below.

[0069] Size Exclusion Chromatogaph:

[0070] SEC was used to assess the level of high molecular weight (HMW)species (aggregate that formed) in the samples during storage. Lowmolecular weight (LMW) species are better resolved by dSEC and that datacan be found in the next section. FIG. 1 shows the SEC data for thesamples stored at 2-8° C. and FIG. 2 shows the SEC data for samplesstored under accelerated conditions of 37° C.

[0071] Data was also collected for samples stored at 30° C. (data notshown) and the levels of HMW species were intermediate to those seen at2-8° C. and 37° C. During storage for 1 year at 2-8° C., aggregatelevels remained stable, or increased less than 0.6% in the worst casefor lot A. No significant increases in aggregate were seen duringstorage at 2-8° C. Under accelerated conditions during storage at 37°C., aggregate levels in lot 1 increased to 19% during 12 months, and to14% and 12% in lot A and B, respectively. The slope of the lines werevery similar, showing that the molecules aggregated at the same rate,and that the differences between lots A-D and lot 1 are due to theinitial levels of aggregate, higher in lot 1 than lots A-D. For lot B,there was no difference between samples stored in a vial at −70° C., ina syringe at −70° C., in a syringe at −20° C., or in a syringe afterthermal treatment and storage at −20° C. (data not shown). All valueswere within 0.4% of the −70° C. vial control (and the time 0 value)after 12 months of storage.

[0072] Denatured Size Exclusion Chromatography:

[0073] Denatured SEC (dSEC) quantitation of the low molecular weight(LMW) species is shown in FIG. 3 for samples stored at 2-8° C., and FIG.4 for samples stored at 37° C. Lots A-D and lot 1 were analyzed by dSECafter storage for up to 1 year at 2-8° C., but lots C and D were notanalyzed past 6 months of storage under the accelerated conditions of37° C. During storage at 37° C., lot 1 and lots A, B, and C showedsimilar breakdown, while lot D showed higher breakdown during heatstressing than lot 1 and the other lots. The similarity in lots A and Band lot 1 was also seen during storage at 30° C. (data not shown), withlevels of breakdown intermediate to that seen at 2-8° C. and 37° C. Forlot B, there was no difference between samples stored in a vial at −70°C., in a syringe at −70° C., in a syringe at −20° C., or in a syringeafter thermal treatment and storage at 20° C. (data not shown). Allvalues after 12 months of storage at −70° C. and −20° C. were within0.7% of each other and the time 0 value.

[0074] During storage at 30° C. (data not shown), the %LMW by dSEC forlot A tracks well with lot B, although both lots show slightly higherlevels of breakdown than lot 1 at this temperature. Lot D shows higherlevels of LMW species at both 2-8° C. and 37° C. at all timepoints,including time 0. The breakdown products in lot D appear to be larger insize than is typically seen by dSEC analysis of stressed TNFR:Fcsamples. These different species are seen after storage at both 2-8° C.and 37° C.

[0075] Hydrophobic Interaction Chromatography:

[0076] HIC was used to separate various TNFR:Fc-related species. Peak 1(and an earlier eluting peak denoted as pre-peak 1) has been shown toconsist mainly of low molecular weight species. Peak 2 includes thefolded, intact dimer (active). Peak 3 includes aggregated material andless active dimers.

[0077] HIC peak 1 data are shown in FIG. 5 for samples stored at 2-8° C.and FIG. 6 for samples stored at 37° C. For all lots except lot D,levels of LMW species remains relatively constant (within 1.2% over 12months) for samples stored at 2-8° C. If the average value for the −70°C. samples is used in place of the time 0 value for lot A, the curvesfor all lots except lot D align well. Lot D shows more peak 1 than theother lots, corroborating the high levels of LMW species seen by dSEC.After heat stressing the samples at 37° C. for up to 1 year, lot B andlot 1 show approximately 30% HIC peak 1, whereas lot A showsapproximately 45% HIC peak 1. Lot D showed 47% HIC peak 1 after only 6months of stressing at 37° C.

[0078] As noted above, HIC peak 2 represents the most desired, activespecies. FIGS. 7 and 8 show the % HIC peak 2 for samples stored at 2-8°C. and 37° C., respectively. Although lot 1 starts out at a lowerinitial % peak 2, it retains the level of active species during storagefor 12 months at 2-8° C. Lots A, B, and C also retain active speciesduring the 12 months of refrigerated storage. Under acceleratedconditions of 37° C., all lots tested lose HIC peak 2 during storage.

[0079] HIC peak 3 levels remained essentially constant during 1 year ofstorage at 2-8° C. (FIG. 9). Variation in % peak 3 for all lots rangedbetween 1 and 3%, well within the error of integration. For lots A, B, Cand D, HIC peak 3 does not show baseline resolution, introducing morevariability in integration. For lot 1, the peak is more clearly defined.After storage at 37° C., the HIC peak 3 levels in lot 1 are morevariable, but remain fairly constant, except for a possible increase at12 months (FIG. 11). Between lots A-D, no clear differences were seenafter storage at 37° C., except for at 12 months, where lot B shows anincrease in HIC peak 3 level.

[0080] Sodium DodecylSulfate-PolyAcrylamide Gel Electrophoresis:

[0081] SDS-PAGE analysis of samples stored for 12 months at −70° C.,−20° C., 2-8° C., 30° C., and 37° C. was performed. Lot A had anincrease in bands associated with both a ˜50 kD and ˜34 kD breakdownfragment after storage at 2-8° C. for 1 year. At elevated temperatures,extensive degradation was seen, with many small molecular weight bandsshowing increased intensities.

[0082] Lot 1 showed no change after 1 year of storage at 2-8° C., butshowed increased ˜50 kD and ˜34 kD breakdown fragment after 1 year at 30and 37° C. Lot B showed no changes during storage for 12 months at −70°C. (vial or syringe) or in syringes at −20° C., with or without thermaltreatment to eliminate supercooling. After 12 months at 2-8° C.,however, bands corresponding to both the ˜50 kD and ˜34 kD breakdownfragment fragments showed increased intensity. Storage at 30 or 37° C.for 1 year resulted in breakdown, with many small molecular weight bandsin addition to the previously discussed ˜50 kD and ˜34 kD breakdownfragment.

[0083] Lots C and D were analyzed after storage for 12 months at −70° C.and 2-8° C. Lot 1, and lots B, C and D are were analyzed after storagefor 12 months at −70° C. and 2-8° C. and showed similar patterns ofdegradation as noted above.

[0084] Binding and Bioactivity:

[0085]FIG. 11 shows the binding activity data derived from an ELISA, forlots A-D and lot 1 stored for 6 and 12 months at −70° C., 2-8° C., 30°C., and 37° C. The error bars on the −70° C. samples indicate +/−30%.Only values outside of these error bars will be considered significantdue to assay variability. Lots A and B retained full binding activityafter 6 months at 2-8 and 30° C., but at 12 months, only the samplesstored at 2-8° C. maintained full binding activity. Lot 1 was able tomaintain full activity for up to 12 months after storage at both 2-8 and30° C., despite showing LMW levels of 13.6% (by dSEC; data not shown)and 8% HMW (by SEC; data not shown) after 1 year at 30° C. Lots C and Dalso retained full binding activity after 1 year of storage at 2-8° C.,despite higher levels of breakdown products seen in lot D by dSEC andHIC.

[0086] An example of a TNFR:Fc bioassay is to inhibit the negativegrowth response of a cell line to human TNF-alpha. The presence ofTNF-alpha inhibits the cells from growing through induction ofapoptosis. The presence of biologically active soluble rhuTNF receptor(TNFR:Fc) specifically neutralizes TNF-alpha in a dose-dependent manner.A TNFR reference standard, control, and samples are added and titratedin a 96 well microtiter plate format. A known concentration of cells isadded to each well followed by addition of TNF-alpha. After anincubation period, non-adherent cells are removed by gently washing withphosphate buffered saline (PBS) and the remaining cells are stained.After an incubation period, each well is read. The units of each wellare directly proportional to the specific activity of TNFR. The resultsfor the bioactivity assay (FIG. 12) corroborate the binding assay data.

CONCLUSIONS

[0087] Lots B and C formulated in a liquid phosphate formulation (25 mMphosphate, 25 mM L-arginine, 98 mM NaCl, 1% sucrose, pH 6.2) were shownto be as stable as lot 1 in the same formulation for 1 year at −70° C.or 2-8° C. Lots A-D showed less aggregation than lot 1, and wereequivalent in terms of breakdown into lower molecular weight species(less than 4% LMW by dSEC at 12 months). Both lot 1 and the lots A-Dshowed increased breakdown and aggregation at elevated temperatures of30 and 37° C., but the lots that performed equal to lot 1 for up to oneyear at 2-8° C. showed equivalence to lot 1 during heat stressing for 1year. Lot D was shown to be less stable in the accelerated assay withhigh levels of low molecular weight breakdown products.

[0088] The data from the accelerated stability testing at 30 and 37° C.corresponds with the long-term stability at 2-8° C., and hence providesa method to accelerate the testing of the long-term stability of aformulated polypeptide at low temperatures without requiring a long-termstability assessment. Samples of lot B stored frozen in syringes at −70°C. and −20° C. showed similar stability to samples stored frozen in avial at −70° C., supporting the use of an embodiment of a pre-filledsyringe stored frozen until delivery to the patient.

[0089] Equivalents and References

[0090] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0091] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

What is claimed is:
 1. A pharmaceutical composition that is a stableaqueous formulation comprising an Fc domain containing polypeptide andan aggregation inhibitor, wherein the aggregation inhibitor isL-arginine.
 2. The composition of claim 1, further comprising a buffer.3. The composition of claim 2, wherein the buffer is selected from thegroup consisting of sodium phosphate, histidine, potassium phosphate,sodium or potassium citrate, maleic acid, ammonium acetate,tris-(hydroxymethyl)-aminomethane (tris), acetate and diethanolamine. 4.The composition of claim 3, wherein the L-arginine is at a concentrationof from about 10 mM to about 100 mM.
 5. The composition of any one ofclaims 1, 2, 3 or 4, further comprising a tonicity modifier.
 6. Thecomposition of claim 5, wherein the tonicity modifier is selected fromthe group consisting of arginine, cysteine, histidine, glycine, sodiumchloride, potassium chloride, sodium citrate, sucrose, glucose andMannitol.
 7. The composition of claim 6, wherein the tonicity modifieris sodium chloride.
 8. The composition of any one of claims 1, 2, 3, or4, further comprising an excipient.
 9. The composition of claim 6,further comprising an excipient.
 10. The composition of claim 7, furthercomprising an excipient.
 11. The composition of claim 8, wherein theexcipient is selected from the group consisting of sucrose, lactose,glycerol, xylitol, sorbitol, Mannitol, maltose, inositol, trehalose,glucose, bovine serum albumin (BSA), human SA or recombinant HA,dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine,gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC),polyethylene glycol, ethylene glycol, glycerol, dimethysulfoxide (DMSO),dimethylformamide (DMF), proline, L-serine, sodium glutamic acid,alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyricacid, Tween-20, Tween-80, SDS, polysorbate, polyoxyethylene copolymer,potassium phosphate, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, zinc ions,copper ions, calcium ions, manganese ions, magnesium ions, CHAPS,sucrose monolaurate, and 2-O-beta-mannoglycerate.
 12. The composition ofclaim 11, wherein the excipient is sucrose.
 13. A stable pharmaceuticalcomposition comprising about 10 mg/ml to about 100 mg/ml TNFR:Fc,L-arginine, sodium phosphate, sodium chloride and sucrose.
 14. Thecomposition of claim 13, wherein L-arginine is about 10 mM to about 75mM.
 15. The composition of claim 13, wherein sodium phosphate is about 5mM to about 100 mM.
 16. The composition of claim 13, wherein sodiumchloride is about 5 mM to about 200 mM.
 17. The composition of claim 13,wherein sucrose is about 0.5% to about 1.5%.
 18. The composition ofclaim 13, wherein pH is about 5.5 to about 7.8.
 19. The composition ofclaim 13, having 25 mg/ml TNFR:Fc, 25 mM L-arginine, 25 mM sodiumphosphate, 98 mM sodium chloride, 1% sucrose at pH 6.2.
 20. Thecomposition of any one of claims 1, 13 or 19, wherein the composition isliquid.
 21. The composition of claim 20, wherein the composition isfrozen.
 22. A method of formulating a composition comprising combiningisolated TNFR:Fc with L-arginine.
 23. The method of claim 22, furthercomprising the steps of combining a buffer, a tonicity modifier, and anexcipient with the composition.
 24. The method of claim 22 or 23,wherein the composition is liquid.
 25. A kit comprising a compositioncomprising an Fc domain containing polypeptide and L-arginine, andinstructions for use of said composition.
 26. The kit of claim 25,wherein the composition is liquid.
 27. The kit of claim 25 or 26,wherein the composition is stored in a pre-filled sterile syringe. 28.The kit of claim 27, wherein the syringe is stored at about −20° C. toabout −70° C.
 29. A method of treating a mammal in need thereofcomprising administering a therapeutically effective amount of thepharmaceutical composition of any one of claims 4 or
 13. 30. A method ofaccelerated stability testing of an Fc domain containing polypeptide ina pharmaceutical composition, wherein the composition comprisesL-arginine, the steps of the method comprising storing the compositionat 37° C. and measuring the stability of the polypeptide after at leastone month at 37° C.